ADP-Ribosylation Factors (ARFs) regulate coat proteins, which act as the core cellular machinery in coupling vesicle formation and cargo sorting for vesicular transport. The ARF small GTPases are, in turn, regulated by guanine nucleotide exchange factors (GEFs) that catalyze ARF activation and GTPase-activating proteins (GAPs) that catalyze ARF deactivation. In the last funding period, we have been studying how three ARF regulators act in endocytic transport. First, we have identified an ARF GAP, known as ACAP1, to act as the core adaptor in a novel clathrin coat complex for endocytic recycling. We have also found that ACAP1 participates in both constitutive and regulated recycling, with its cargo binding explaining how both types of transport can be accomplished. Thus, we propose to further elucidate how ACAP1 acts in cargo sorting by taking combined biochemical and structural approaches. Second, we have identified Grp1 as the GEF that acts on ARF6 for the endocytic recycling of the glucose transporter type 4 (glut4), a process that is regulated by insulin. Thus, we propose to take combined morphologic and biochemical approaches to elucidate how this GEF may be targeted by insulin-induced signaling that regulates glut4 recycling. Third, we have found that ARFGAP1 is required for a subset of clathrin AP2-dependent transport, as defined by the endocytosis of transferrin receptor. Thus, we will further elucidate this role, examining how ARFGAP1 participates in cargo sorting and vesicle formation and also interrogating the role of its GAP activity. We anticipate that these results will shed key insights into mechanisms of endocytosis and recycling. Moreover, because defects in mechanisms that govern these transport pathways can cause and/or contribute to cardiovascular, immunologic, neurologic and oncogenic diseases, we further anticipate that our results will have broad ramifications to understanding and treating human diseases. PUBLIC HEALTH RELEVANCE: The function of proteins is critically regulated by their localization. This localization is achieved in part by transport pathways that act as highways within the cell. We propose to understand how key components of these pathways function in regulating the distribution of proteins on the cell surface. This understanding will be broadly applicable to understanding human diseases, as many are now appreciated to arise because of defects in protein transport within the cell.